EVIDENCE FOR 2 CATALYTIC SITES IN THE FUNCTIONAL UNIT OF H-ATPASE FROM HIGHER-PLANTS()

We investigated the nature of the complex ATP activation kinetics of p lant H+-ATPases. To this aim we analyzed that activation in three isol ated isoforms (AHA1, AHA2, and AHA3) of H+-ATPase from Arabidopsis tha liana. The isoforms were obtained by heterologous expression in endopl asmic reticulum of yeast. ATP stimulation was always with low affinity (K-0.5 between 500 and 1800 mu M). In addition, the curves were not M ichaelian and displayed positive cooperativity. Detailed studies with AHA2 showed that (a) enzyme solubilized with lysophosphatidylcholine e xhibited Michaelian behavior even in the presence of soybean lecithin liposomes free of enzyme, (b) solubilized enzyme incorporated into the same liposomes displayed two-site kinetics with negative cooperativit y, and (c) enzyme partially digested with trypsin lost the C-terminal portion of the molecule. Under this condition the ATP activation kinet ics was Michaelian or had a slight negative cooperativity and the K-0. 5(ATP) was reduced 3-fold. These data suggest that the functional unit of the H+-ATPase has two catalytic ATP sites with variable cooperativ ity and kinetics competence of the E(ATP) and E(ATP)2 complexes. Such variability is likely modulated by the association of the enzyme with membrane structures and by a regulatory domain in the C terminus of th e enzyme molecule.